Botulinum toxins (BoNT) and tetanus toxin (TeNT) are the most toxic proteins for humans. Based upon this potency and concern for intentional release, BoNTs are classified by the CDC as Tier 1, Category A Select Agents. There are no approved vaccines or therapies against botulism. Conversely, BoNT is an effective therapy for numerous human neurological diseases to treat dystonia, and neuromuscular contractions/spasms; and tetanus toxoid is a potent vaccine against tetanus and a platform for conjugate vaccines to immunize against several bacterial pathogens. Despite this toxicity and extensive use in human therapies and vaccines, how these toxins intoxicate neurons to elicit their unique spasticity remains undefined. BoNT and TeNT are AB single chain toxins with an N-terminal Light Chain (LC) and C-terminal Heavy Chain (HC), which includes a Translocation domain (HCT) and Receptor binding domain (HCR). During the previous funding period, a novel assay was developed to measure LC translocation into the cytosol of neurons, the least understood step in toxin action. BoNT was shown to enter neurons via synaptic vesicles upon membrane depolarization, while TeNT entered endosomes independent of synaptic vesicle cycling. A recombinant, non-toxic full length TeNT was engineered and used to show that regions outside the HCR domain targeted TeNT to endosomes, which changes our perception that the interaction between toxins and host cell receptors is solely responsible for cell entry. W propose a novel model termed the BoNT Default Pathway to describe the unique molecular steps for BoNT and TeNT entry in neurons. In this model, BoNTs bind neurons solely through interactions between the HCR and host receptors and enter neurons during synaptic vesicle cycling. In contrast, while TeNT also binds to host receptors via HCR interactions, TeNT also binds to growth factor receptors, such as p75NTR, which traffic TeNT into signaling endosomes to retrograde traffic within a neuron. This renewal will utilize biochemical, cellular, and moleculr techniques to define the mechanisms responsible for this unique entry and trafficking of BoNT and TeNT into neurons. Our hypothesis is that BoNT and TeNT enter neurons through unique pathways, but share common mechanisms to deliver LC into the cytosol of neurons. The three specific aims will determine how BoNT enters synaptic vesicles, how TeNT enters signaling endosomes, and will characterize Light Chain translocation into host cells. Completion of these studies will resolve how BoNT and TeNT enter neurons to elicit the spastic paralysis of tetanus and the flaccid paralysis of botulism. These studies will impact the development of vaccines and therapies against botulism and the next generation of tetanus toxin as a conjugate vaccine carrier. The outcomes of these studies may also allow the engineering of these neurotoxins as probes and therapies for complex human neurological diseases that are refractive to medical intervention.